Fruit, vegetables, and plants are all susceptible to attack by fungi, resulting in loss of crops, decreased shelf-life of produce, and ultimately higher costs for consumers. Many fungi are known pathogens in several diseases which harm or destroy crops worldwide. Examples of such fungi include those belonging to the genera Rhizoctonia, Pythium, Gaeumannomyces, and Fusarium. 
For a number of years, it has been known that various microorganisms exhibit biological activity useful in controlling plant diseases. Although progress has been made in the field of identifying and developing biological pesticides for controlling plant diseases of agronomic and horticultural importance, most of the pesticides in use are still synthetic compounds. Many of the chemical fungicides are carcinogenic agents and, therefore, toxic to wildlife and other non-target species. In addition, pathogens may develop resistance to chemical pesticides. In fact, the fungicides, considered the major weapon in combating plant diseases, are often ineffective and pose hazards to humans and the environment. Biological control offers an attractive approach as compared with synthetic chemical fungicides. Biopesticides (living organisms and the compounds which are naturally produced by these organisms) can be safer, more biodegradable, and less expensive to develop. In addition, they are highly desired for integrated pest management programs in agriculture, public health, and urban settings.
The agricultural use of Bacillus megaterium has been reported for disease control in rice and cotton inhibition as seed treatment but not as foliar sprays. U.S. Pat. No. 5,403,583 discloses a Bacillus megaterium, ATCC 55000, and a method to control the fungal plant pathogen Rhizoctonia solani as seed treatment. Islam and Nandi also disclosed a Bacillus megaterium with antagonism to Drechslera oryzae, the causal agent of rice brown spot (Journal of Plant Diseases and Protection. 92(3): 241–246 (1985) and a Bacillus megaterium with in vitro antagonism against Drechslera oryzae, Alternaria alternata and Fusariun roseum (Journal of Plant Diseases and Protection. 92(3): 233–240 (1985). They mentioned three components in the culture filtrate. The most active antibiotic was highly soluble in water and methanol with a UV peak at 255 nm and a shoulder at 260 nm, which proved to be a polyoxin-like lipopeptide. And, Cook (Proceedings Beltwide Cotton Production-Mechanization Research Conference, Cotton Council, Memphis, p. 43–45 (1987) disclosed the use of a suspension of Bacillus megaterium to reduce the number of plants killed by Phymatotrichum omnivorum, a cause of cotton root rot. Antibiotic production of B. megaterium has also been recorded by Berdy (CRC Handbook of Antibiotic Compounds, Vols. I–XIV. CRC Press, Inc. Boca Raton. Fla. 1980–87), who reported the production of low-mammalian toxic peptide antibiotics such as ansamitocin-PDM-O, bacimethrin, megacin, pentapeptide, and homopeptides.
U.S. Pat. No. 5,494,819 describes a pure culture of Pantoea agglomerans having all of the characteristics of FERM BP-3511 which is identified by growth, morphology, physiology, utilization of carbon sources and various specific enzymatic tests involving enzymes as lysine decarboxylase, arginine dihydroxylase, phenylalanine deaminase and ornithine decarboxylase. In addition, the disclosed pure culture of Pantoea agglomerans is characterized by the required production of lipopolysaccharides to which the inventors attribute an immunity-stimulating activity. In other words, according to this document, Pantoea agglomerans is used to obtain substances to be used in pharmaceuticals.
U.S. Pat. No. 5,766,926 discloses a method comprising the steps of applying to the pulpwood or pulp substrate a bacterial inoculum of at least one of the species selected from the group consisting of Pseudomonas fluorescens, Pantoea (Enterobacter) agglomerans, Bacillus cereus, and Xanthomonas campestris and maintaining the substrate under conditions which allow bacterial growth for a time sufficient to effect a reduction in the resin component of the substrate by the bacteria. It is mentioned that the source of the Pantoea (Enterobacter) agglomerans isolate used in the method, identified by the NRRL Accession No.B21509, is Brazil.
It is known that the genus Fusarium contains species which may cause diseases of wither and blight that occur during the growth of plants and damages not only the host but also other kinds of plants. It is supposed that fusaric acid is the principal agent that brings about these diseases. Fusaric acid (5-n-butylpicolinic acid) is known to be a non-specific toxin which is produced by the metabolism of almost all plant pathogenic Fusarium fungi (Wood, R. K. et al. 1972. “Phytotoxins in plant diseases”. Academic Press. New York; Durbin, R. D. 1982. “Toxins in plant diseases”. Academic Press. New York). In the document EP 257 756, referring to the prevention of Fusarium diseases and microorganisms therefor, the inventors proposed to prevent such diseases by using microorganisms belonging to the genera Cladosporium and Pseudomonas which decompose fusaric acid. EP 441 520 relates also to detoxifying fusaric acid microorganisms, and Klebsiella oxytoca HY-1 (FERM BP-3221) is particularly mentioned.
In the document WO 92018613, it is suggested to control plant diseases caused by fungi of the genera Rhizoctonia, Pythium, and Fusarium by using a new strain of Pseudomonas fluorenscens, a seed or soil treatment but not foliar sprays.
WO 9905257, referring to biocontrol for plants with Paenibacillus macerans, Pseudomonas putida, and Sporobolomyces roseus, describes the use of isolates of these microorganisms to impart pathogen protection to plants, particularly against plant diseases caused by fungi, such as Fusarium oxysporum, Fusarium graminearum, Fusarium monilforme, Cochliobolus sativus, Collectotrichum graminicola, Stagonospora nodorum, Stagonospora avenae, Stenocarppela maydis, and Pyrenophora tritici-repentis. In this case, pathogen protection was achieved by either seed treatment or foliar sprays.
Fusarium graminearum Schw. (Teleomorph=Gibberella zeae Schw. Petch.) is the Fusarium species most frequently responsible for scab of wheat and barley in Brazil. This disease, also known as Fusarium Head Blight (FHB), is responsible for major losses which vary from 10% (see Luz, W. C. da. 1984. “Yield losses caused by fungal foliar wheat pathogens in Brazil”. Phytopathology. 74:1403–1407); to 54% (Picinini, E. C. and Fernandes, J. M. C. 1994. “Controle quimico da Gibberella zeae em trigo pelo uso de fungicidas inibidores da síntese do ergoterol”. Fitopatol. Brasileira 19 (Supl.):273). At present, available and affordable control measures, such as resistant varieties, cultural practices, and foliar fungicides, are only partially effective.
Only modest levels of resistance have been deployed in cultivars in commercial fields; the most widely grown cultivars are often most susceptible. Furthermore, the benefit of crop rotation as a control measure is reduced by the wide host range of the pathogen, especially on grasses (Costa Neto, J. P. da. 1976. “Lista de fungos sobre gramíneas (capins e cereais) no Rio Grande do Sul”. Revista da Faculdade de Agronomia. UFRGS. 1:43–78; Luz, W. C. da. 1982. “Diagnose das Doencas da Cevada”. Passo Fundo—EMBRAPA-CNPT, 24p. (Circular Técnica no. 2)). Treatment with foliar fungicides remains the most important (Picinini and Fernandes, 1994) and recommended (Reunião da Comissão Sul-Brasileira de Pesquisa de Trigo, 2000) tool for reducing scab in Brazil, despite its shortcomings as a control measure. The use of certain effective fungicides has been restricted in some countries because application at late developmental stages, that is, during heading and flowering, can result in chemical residues in the harvested grain. Biological control is an additional strategy that may eventually play an important role in an integrative approach to scab management of cereals.
Screening of microorganisms to control wheat scab was initiated in Brazil in the 1980's (Luz, W. C. da. 1988. “Biocontrol of fungal pathogens of wheat with bacteria and yeasts”. Page 348 in: 5th International Congesss of Plant Pathology, Kyoto, Japan. (Abstr.)). At the beginning, over 300 bacteria and yeasts isolated from wheat were screened in vitro against F. graminearum. This work was followed by that of Perondi et al. (Perondi, N. L., Thomas, R. and Luz, W. C. da. 1990. “Antagonistas potenciais de Fusarium graminearum”. In: Anais do 2o Simpósio de Controle Biológico, Brasilia, D. F., p. 128. (Abstr.); Perondi, N. L., Thomas, R. and Luz, W. C. da. 1990. “Controle microbiano da giberela do trigo em campo”. In: Anais do II Simpósio de Controle Biológico, Brasilia, D F. P.129(Abstr.); Perondi, N. L., Luz, W. C. da. and Thomas, R. 1996. “Controle microbiológico da giberela do trigo”. Fitopatol. Brasileira 21:243–249) in which microbial strains were tested for their antagonistic action against the pathogen. Potential antagonists were selected by the funnel method (Luz, W. C. da. 1990. “Microbiological control of Bipolaris sorokiniana ‘in vitro’”. Fitopatol. Brasileira 15:246–247) which compared the effect of individual test organisms on the radial growth of F. graminearum. Promising isolates were further tested in the greenhouse and in the field for their ability to control wheat scab. Individual bioprotectants significantly diminished the severity of the disease under field conditions, raising the yield of wheat between 7 and 31% when compared to non-treated plants.
Besides the selection of the bioprotectants, it is important to overcome several difficulties related to constraints on their application to the ears of wheat and barley at flowering such as the timing of application, inoculation technology, physiological state of the organisms, spike colonization, survival of the organisms under the harsh environmental conditions, variability of biocontrol from year to year, fermentation, formulation, and storage. The partial control of any tactics to protect against FHB up to this moment indicates that the integration of protection measures would provide the best disease management.
From 1988 up to now, thousands of microorganisms have been tested for scab control. Some workers have been investigating antagonists to control FHB (Khan, N. J., Schisler, D. A., Boehm, M. J., Lipps, P. E., Slininger, P. J. and Bothast, R. J. 1998. “Biological control of scab of wheat incited by Giberella zeae”. Pages 45–46 in: Proceedings of the 1998 National Fusarium Head Blight Forum, Michigan State University, University Printing, East Lansing. Mich.; Khan, N. J., Schisler, D. A., and Boehm, M. J. 1999. USDA-ARS, Ohio State University cooperative research on biologically controlling Fusarium Head Blight: 2. Influence of pathogen strain, inoculum spray sequence and inoculum spray time. Pages 56–59 in: Proceedings of the 1999 National Fusarium Head Blight Forum, Michigan State University, University Printing, East Lansing, Mich.; Boehm, m. J., Khan, N. J., and Schisler, D. A. 1999. USDA-ARS, Ohio State University cooperative research on biologically controlling Fusarium Head Blight: 3. Field testing of antagonists. Pages 45–48 in: Preceedings of the 1999 National Fusarium Head Blight Forum, Michigan State University, University Printing, East Lansing, Mich.; Luo, Y. and Bleakley, B. 1999. “Biological control of Fusarium Head Blight (FHB) of wheat by Bacillus strains”. Pages 78–81 in: Proceedings of the 1999 National Fusarium Head Blight Forum, Michigan State University, University Printing, East Lansing, Mich.; Schisler, D. A., Khan, N. J., and Boehm, M. J. 1999. USDA_ARS, Ohio State University cooperative research on biologically controlling Fusarium Head Blight: 1. Antagosist selection and testing on durum wheat. Pages 78–81 in: Proceedings of the 1999 National Fusarium Hrad Blight Forum, Michigan State University, University Printing, East Lansing, Mich.; Stockwell, C. A., Luz, W. C. da., and Bergstrom, G. C. 1997. “Biocontrol of wheat scab with microbial antagonists”. Phytopathology 87:S94.(Abstr.); Stockwell, C. A., Bergstrom, G. C., and Luz, W. C. da.1999. “Selection of microbial antagonists for biological control of Fusarium Head blight of wheat”. Pages 82–84. in: Proceedings of the 1999 National Fusarium Head Blight Forum, Michigan State University, University Printing, East Lansing, Mich.; Stockwell, C. A., Bergstrom, G. C., and Luz, W. C. da. 2000. “Identification of bioprotectants for biological control of Gibberella zeae” in:. Proceedings of FHB Forum), under greenhouse or field conditions. Some strains have reduced the FHB severity and significantly reduced vomitoxin contamination in grains (Stockwell et al., 1997, 2000). Table 1 illustrates the chronology of researches on the biocontrol of FHB.
TABLE 1Chronology of works done on biocontrol of Fusarium Head Blight ofwheatLITERATUREBIOPROTECTANTSLus, W.C. da, 1988Bacteria, YeastPerondi; N.L., Luz, W.C. da & Thomas, R,Bacillus subtilis1990 a, 1990 b, 1996Bacillus spp.Pseudomonas fluorescensSporobolomyces roseusStockwell, C.A; Luz, W.C. da, andPaenibacillus maceransBergstrom, G.C. 1997Pseudomonas putidaSporobolomyces roseusKhan, N.I., Scisler, D.A.. Bochm, M.J,Bacillus spp.Lipps, P.E., Slininger, P.J. and Bothast,R.J., 1998Boehm, M.J., Khan., N.J., and Schisler,Yeast, Bacillus sp.D.A, 1999Khan, N.J., and Schisler, D.A., andYeast, Bacillus sp.Boehm, M.J., 1999Luo, Y. & Bleakely, B. 1999Bacillus spp.Schisler, D.A., Khan, N.J. and Boehm,Bacillus spp.M.J. 1999Stockwell, C.A., Bergbstrom, G.C. andPaenibacillus maceransLuz, W.C. da. 1999Pseudomonas putidaSporobolomyces roseusStockwell, C.A., Bergstrom, G.C. andPaenibacillus maceransLuz, W.C. da., 2000Bacillus spp.